Bottom Line:
CB2 genome revealed various rearrangements had occurred in comparison to what was observed in 3As and K12 and over 20 genomic islands (GEIs) were found in each of these three genomes.We performed a detailed comparison of the two arsenic-related islands found in CB2, carrying the genes required for arsenite oxidation and As resistance, with those found in K12, 3As, and five other Thiomonas strains also isolated from Carnoulès (CB1, CB3, CB6, ACO3 and ACO7).Our results suggest that these arsenic-related islands have evolved differentially in these closely related Thiomonas strains, leading to divergent capacities to survive in As rich environments.

ABSTRACTAcid mine drainage (AMD) is a highly toxic environment for most living organisms due to the presence of many lethal elements including arsenic (As). Thiomonas (Tm.) bacteria are found ubiquitously in AMD and can withstand these extreme conditions, in part because they are able to oxidize arsenite. In order to further improve our knowledge concerning the adaptive capacities of these bacteria, we sequenced and assembled the genome of six isolates derived from the Carnoulès AMD, and compared them to the genomes of Tm. arsenitoxydans 3As (isolated from the same site) and Tm. intermedia K12 (isolated from a sewage pipe). A detailed analysis of the Tm. sp. CB2 genome revealed various rearrangements had occurred in comparison to what was observed in 3As and K12 and over 20 genomic islands (GEIs) were found in each of these three genomes. We performed a detailed comparison of the two arsenic-related islands found in CB2, carrying the genes required for arsenite oxidation and As resistance, with those found in K12, 3As, and five other Thiomonas strains also isolated from Carnoulès (CB1, CB3, CB6, ACO3 and ACO7). Our results suggest that these arsenic-related islands have evolved differentially in these closely related Thiomonas strains, leading to divergent capacities to survive in As rich environments.

pone.0139011.g004: CB2 capacity to oxidize arsenite.Concentrations of As(III) (squares) and As(V) (triangles) are shown for CB2 cells grown in m126 medium in the presence of 2.66 mM of As(III) (full symbols) and m126 medium in the presence of 2.66 mM of As(III) and supplemented with glucose (hollow symbols). Error bars indicate standard deviations of triplicate cultures. No As(III) oxidation was observed in abiotic controls (data not shown).

Mentions:
The CB2, K12, and 3As Thiomonas genomes harbor an arsRICB operon, sharing more than 97% of similarity at the nucleotide level, that is not located in a GEI but in a very conserved region of these three genomes. Both CB2 and 3As have an additional arsRDACB operon with a shared 91% identity at the nucleotide level, located in a GEI in both genomes. In CB2, this operon is found on RGP19, directly upstream from an aio locus, while in 3As, it is located on The ThGEI-O described previously [16]. In CB2, two versions of the aio operon, sharing less than 90% identity at the nucleotide level, are located in two distinct GEIs (referred to from now on as “arsenic genomic islands”) (Fig 3). One copy is encoded in RGP19 of CB2, while the second is located in RGP10. In contrast, a single aio operon is found in 3As [16] and in K12 (this work). The CB2 AioA and AioB proteins encoded by the operon located on RGP19 both share 100% identity with the proteins encoded by 3As, and 84% and 88% identity with those of K12, respectively (Table 1). However, the AioA and AioB encoded by the operon on RGP10 share 82% and 89% identity with the 3As proteins and 87% and 98% identity with those of K12 (Fig 3 and Table 1). These observations revealed than one copy of the aio locus and associated genes of CB2 (located on RGP19), were more related to those in 3As, whereas the second copy of the aio and associated genes in CB2 (located on RGP10), were more related to those found in the unique aio locus of K12 (Fig 3, Table 1 and S2 Fig). To test if the genes are functional in CB2, we assessed the extent of As(III) oxidation, and found that more than 60% of As(III) was oxidized to As(V) within 48 h, followed by a slow reduction of As(V) during the stationary growth phase (Fig 4). In other Thiomonas isolates, where arsenite oxidation is involved in energy production, this activity was reduced in the presence of a preferred substrate. In our work, the addition of glucose to m126 caused a severe inhibition of As(III) oxidation in CB2 (Fig 4), suggesting that CB2 may use either arsenite or glucose as an electron donor and the inhibition of the arsenite oxidation could be due to a catabolite repression. Finally, we investigated if both copies of the aio genes were expressed in two growth conditions. First, RNAseq was performed on cells grown in conditions favoring biofilm formation (without shaking) and revealed that the expression of the ars and the aio genes (two copies of each operon), and also other genes from these two islands, are induced by As(III) (S4 Table). Second, a Western blot was performed to determine if both copies were synthetized during aerobic growth. In shaken liquid cultures, we observed that indeed, the two aioA genes were expressed only in the presence of As(III) (S3 Fig), as was found previously in 3As [18]. Altogether, these experiments demonstrated that both CB2 aioAB operons are functional.

pone.0139011.g004: CB2 capacity to oxidize arsenite.Concentrations of As(III) (squares) and As(V) (triangles) are shown for CB2 cells grown in m126 medium in the presence of 2.66 mM of As(III) (full symbols) and m126 medium in the presence of 2.66 mM of As(III) and supplemented with glucose (hollow symbols). Error bars indicate standard deviations of triplicate cultures. No As(III) oxidation was observed in abiotic controls (data not shown).

Mentions:
The CB2, K12, and 3As Thiomonas genomes harbor an arsRICB operon, sharing more than 97% of similarity at the nucleotide level, that is not located in a GEI but in a very conserved region of these three genomes. Both CB2 and 3As have an additional arsRDACB operon with a shared 91% identity at the nucleotide level, located in a GEI in both genomes. In CB2, this operon is found on RGP19, directly upstream from an aio locus, while in 3As, it is located on The ThGEI-O described previously [16]. In CB2, two versions of the aio operon, sharing less than 90% identity at the nucleotide level, are located in two distinct GEIs (referred to from now on as “arsenic genomic islands”) (Fig 3). One copy is encoded in RGP19 of CB2, while the second is located in RGP10. In contrast, a single aio operon is found in 3As [16] and in K12 (this work). The CB2 AioA and AioB proteins encoded by the operon located on RGP19 both share 100% identity with the proteins encoded by 3As, and 84% and 88% identity with those of K12, respectively (Table 1). However, the AioA and AioB encoded by the operon on RGP10 share 82% and 89% identity with the 3As proteins and 87% and 98% identity with those of K12 (Fig 3 and Table 1). These observations revealed than one copy of the aio locus and associated genes of CB2 (located on RGP19), were more related to those in 3As, whereas the second copy of the aio and associated genes in CB2 (located on RGP10), were more related to those found in the unique aio locus of K12 (Fig 3, Table 1 and S2 Fig). To test if the genes are functional in CB2, we assessed the extent of As(III) oxidation, and found that more than 60% of As(III) was oxidized to As(V) within 48 h, followed by a slow reduction of As(V) during the stationary growth phase (Fig 4). In other Thiomonas isolates, where arsenite oxidation is involved in energy production, this activity was reduced in the presence of a preferred substrate. In our work, the addition of glucose to m126 caused a severe inhibition of As(III) oxidation in CB2 (Fig 4), suggesting that CB2 may use either arsenite or glucose as an electron donor and the inhibition of the arsenite oxidation could be due to a catabolite repression. Finally, we investigated if both copies of the aio genes were expressed in two growth conditions. First, RNAseq was performed on cells grown in conditions favoring biofilm formation (without shaking) and revealed that the expression of the ars and the aio genes (two copies of each operon), and also other genes from these two islands, are induced by As(III) (S4 Table). Second, a Western blot was performed to determine if both copies were synthetized during aerobic growth. In shaken liquid cultures, we observed that indeed, the two aioA genes were expressed only in the presence of As(III) (S3 Fig), as was found previously in 3As [18]. Altogether, these experiments demonstrated that both CB2 aioAB operons are functional.

Bottom Line:
CB2 genome revealed various rearrangements had occurred in comparison to what was observed in 3As and K12 and over 20 genomic islands (GEIs) were found in each of these three genomes.We performed a detailed comparison of the two arsenic-related islands found in CB2, carrying the genes required for arsenite oxidation and As resistance, with those found in K12, 3As, and five other Thiomonas strains also isolated from Carnoulès (CB1, CB3, CB6, ACO3 and ACO7).Our results suggest that these arsenic-related islands have evolved differentially in these closely related Thiomonas strains, leading to divergent capacities to survive in As rich environments.

ABSTRACTAcid mine drainage (AMD) is a highly toxic environment for most living organisms due to the presence of many lethal elements including arsenic (As). Thiomonas (Tm.) bacteria are found ubiquitously in AMD and can withstand these extreme conditions, in part because they are able to oxidize arsenite. In order to further improve our knowledge concerning the adaptive capacities of these bacteria, we sequenced and assembled the genome of six isolates derived from the Carnoulès AMD, and compared them to the genomes of Tm. arsenitoxydans 3As (isolated from the same site) and Tm. intermedia K12 (isolated from a sewage pipe). A detailed analysis of the Tm. sp. CB2 genome revealed various rearrangements had occurred in comparison to what was observed in 3As and K12 and over 20 genomic islands (GEIs) were found in each of these three genomes. We performed a detailed comparison of the two arsenic-related islands found in CB2, carrying the genes required for arsenite oxidation and As resistance, with those found in K12, 3As, and five other Thiomonas strains also isolated from Carnoulès (CB1, CB3, CB6, ACO3 and ACO7). Our results suggest that these arsenic-related islands have evolved differentially in these closely related Thiomonas strains, leading to divergent capacities to survive in As rich environments.